Cryptosporidium spp. are ubiquitous waterborne parasites responsible for outbreaks of diarrheal disease worldwide [1]. The infection is self-limiting in immunocompetent individuals, but in immunocompromised individuals the infection can develop into a chronic, debilitating, and sometimes life-threatening disease [2]. In malnourished children, cryptosporidiosis is associated with growth and developmental delays [1]. Currently, there are no vaccines to prevent cryptosporidiosis, and nitazoxanide, the only approved drug in the US [3] is not effective in AIDS patients [4]. Because the parasite cannot be propagated in vitro or genetically manipulated, data that would permit targeted drug design or vaccine development are sorely lacking.Ingested Cryptosporidium oocysts excyst in the intestine, releasing sporozoites that attach to and invade intestinal epithelial cells, initiating the lifecycle [5]. The parasite undergoes two rounds of asexual reproduction, in each cycle producing merozoites that invade new epithelial cells, before entering the sexual cycle and producing oocysts. One research focus has been to identify the antigens on the invasive zoite stages that allow the parasite to attach to and invade the epithelial cell with the goal of preventing these parasite-host cell interactions. The best characterized of the zoite antigens is Cpgp40/15, (also called Cp17, gp15/45/60 or S60) [6][7][8][9][10], a mucin like glycoprotein antigen that is synthesized as a single precursor protein and proteolytically cleaved into the mature glycoproteins, gp40 and gp15 [11]. gp15 is anchored in the sporozoite membrane by a glycosylphosphatidyl inositol (GPI) moiety [12], while the gp40 glycoprotein does not contain any predicted transmembrane domains or GPI anchors and is predicted to be soluble [7]. However, gp40 has been shown to bind intestinal epithelial cells in a dose dependent and saturable manner [7] suggesting that this antigen recognizes a host cell receptor. If this interaction is important for zoite attachment and invasion, then gp40 must have some mechanism of associating with the parasite membrane. In these studies we explore the possibility that gp40 and gp15 associate to form a protein complex capable of linking zoite and host cell surfaces.* Corresponding Authors NEMC Box 041, 750 Washington St, Boston, MA 02111, e-mail: roconnor@tufts-nemc.org; hward@tufts-nemc.org, tel ROC: 617 636 2684, HW: 617 636 7022, fax: 617 636 5292. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. To generate gp40 specific antiserum, recombinant (r)gp40 fusion protein [7] was purified via the His...